Review Immune Responses Induced by mRNA Vaccination in Mice, Monkeys and Humans Alberto Cagigi and Karin Loré * Division of Immunology and Allergy, Department of Medicine Solna, Karolinska Institutet, Karolinska University Hospital, 161 64 Solna, Sweden; [email protected] * Correspondence: [email protected] Abstract: In this concise review, we summarize the concepts behind mRNA vaccination. We dis- cuss the innate and adaptive immune response generated by mRNA vaccines in different animal models and in humans. We give examples of viral infections where mRNA vaccines have shown to induce potent responses and we discuss in more detail the recent SARS-CoV-2 mRNA vaccine trials in humans. Keywords: mRNA vaccine; dendritic cell; protein translation; type I interferon; Th1 polarization; antibody response; SARS-CoV-2 1. Introduction The first successful experimental injections of mRNA that resulted in translated pro- teins were performed in murine muscle cells in vitro in the early 1990s [1]. This encouraged the development of mRNA and also DNA as a new concept of vaccination [2–4]. How- ever, it was not until critical modifications of the mRNA leading to increased stability and translational capacity were introduced that the field of mRNA vaccination started Citation: Cagigi, A.; Loré, K. its real expansion [5]. The interest in mRNA vaccination has been growing substantially Immune Responses Induced by the last decade, and has during the current SARS-CoV-2 pandemic further accelerated as mRNA Vaccination in Mice, Monkeys some of the first vaccine candidates approved for human use are mRNA vaccines (https: and Humans. Vaccines 2021, 9, 61. //www.raps.org/news-and-articles/news-articles/2020/3/covid-19-vaccine-tracker). A https://doi.org/10.3390/vaccines competitive advantage of mRNA vaccines that has been particularly emphasized is that 9010061 the vaccine production and purification processes are similar, despite the encoded antigens. This feature gives prospects of using harmonized protocols for different mRNA vaccines [6]. Received: 23 December 2020 Production of new mRNA vaccines can thereby be initiated immediately once the genomic Accepted: 12 January 2021 Published: 18 January 2021 sequence of a target antigen has been identified. This can result in reduced costs and faster production of new vaccines against emerging infectious diseases, as exemplified by the Publisher’s Note: MDPI stays neutral mRNA vaccines against SARS-CoV-2. Concerns with reproducibility of virus cultures with regard to jurisdictional claims in and protein production in mammalian cells or issues with egg allergies associated with published maps and institutional affil- seasonal influenza vaccines produced in eggs are circumvented by mRNA vaccines. mRNA iations. vaccines, as opposed to DNA vaccines, do not need to be delivered into the cell nucleus via electroporation or other devices, since protein translation from mRNA occurs in the cytoplasm and regular needle injection is sufficient. In addition, there is not a need for vaccine adjuvants, since mRNA itself has the inherent ability to induce a strong innate im- mune response [7,8]. However, the innate immune activation by mRNA vaccines may also Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. result in side effects and limited protein translation followed by weak adaptive immune This article is an open access article responses. Fine-tuning the innate and the adaptive immune responses by modifying the distributed under the terms and mRNA, the mRNA dosage and the formulation strategy is a major area of research. conditions of the Creative Commons Two forms of mRNA vaccines have primarily been studied. The main group consists Attribution (CC BY) license (https:// of (1) the more conventional chemically modified and unmodified sequence-optimized creativecommons.org/licenses/by/ mRNA vaccines and (2) the self-amplifying mRNA vaccines based on virally derived 4.0/). RNA that encodes both the antigen of interest and the proteins enabling RNA vaccine Vaccines 2021, 9, 61. https://doi.org/10.3390/vaccines9010061 https://www.mdpi.com/journal/vaccines Vaccines 2021, 9, 61 2 of 14 replication. The advantage of self-amplifying mRNA vaccines is that a lower dose of mRNA can often be used, which also results in decreased (Toll-like-receptor) TLR recognition and associated innate immune responses. Yet, the conventional mRNA vaccines have progressed the furthest into clinical practice so far. Apart from the important improvements in optimizing the mRNA in both groups of mRNA vaccines for translatability, stability and reactogenicity over the years, major advancements have also been made regarding potent and well-tolerated delivery technologies, such as lipid-based drug delivery systems [9,10]. This is a critical part in the development of mRNA vaccines. Early studies showed that even if the mRNA is stabilized, a large proportion is simply filtered out via the kidneys and urine after administration due to its small size [11]. Significant improvements in the bioavailability of mRNA through formulation have since been made. The majority of mRNA vaccines are currently packaged in biodegradable ionizable lipid nanoparticles (LNPs) consisting of variants of phospholipids, cholesterol and polyethylene glycol (PEG) containing lipids [10,12]. The ionizable lipid is positively charged to form complexes with the negatively charged mRNA for protection of the mRNA and may also help with cellular uptake and endosomal escape [12]. PEG–lipids significantly increase the bioavailability, i.e., time of mRNA in the circulation, which greatly improves the prospects for therapeutic use, but this can be at the expense of reduced transfection efficiency [13]. Modifying the PEG– lipids or cholesterol contents can also be utilized to alter the particle size and morphology of the LNP and in turn influence the trafficking routes after administration and the efficiency of mRNA delivery to cells [10,12–14]. Furthermore, mixing of unsaturated lipids can also improve the uptake by cells [15]. Alternative nanoparticles described are the core-shell structured lipopolyplex (LPP) platform in which mRNA binds to a positively charged protein or polymer to form a dense core structure that is encapsulated in a lipid shell [16]. Here we review the characteristics of reported immune responses induced by different mRNA vaccines in mouse and non-human primate (NHP) models versus humans. Fur- thermore, we speculate on the strengths and weaknesses of mRNA vaccines compared to conventional vaccine platforms. 2. Innate Immune Activation of mRNA Vaccines Live-attenuated viruses are amongst the most successful vaccines in eliciting high and long-lasting protection, as illustrated by the measles vaccine and the yellow fever vaccine that can induce antibody levels that are maintained above the protective threshold for decades [17,18]. This might be due to the fact that attenuated infection and viral replication most closely mimic the characteristics of the natural pathogen which results in elicitation of a stronger immune response. Local inflammation at the injection site and recruitment of antigen presenting cells (APCs) are essential to promoting adaptive cellular T cell responses for eliminating infected cells and humoral (antibody) responses for neutralizing pathogens. However, live vaccines may not be administered to immune compromised individuals because of safety risks [19]. Instead, killed/inactivated, split virion or protein-based vaccines are more suitable for a broader population, but are often poorly immunogenic and require an adjuvant to induce sufficient responses. The by-far most clinically used adjuvant, alum, primarily generates a Th2 response. Other adjuvants, such as Toll-like-receptor (TLR) agonists can shift this balance via, for example, production of type I interferon (IFN), which promotes Th1 responses mimicking the milieu of a viral infection [20]. The type I IFN responses induced naturally by the presence of mRNA and the downstream Th1-polarized responses induced are also characteristic for mRNA vaccines [8,21]. This may make mRNA vaccines particularly suitable for viral infections. In addition, upon vaccination with mRNA, the antigenic protein is produced by host cells similar to a viral infection, which can lead to some degree of MHC class I presentation, even if the mRNA sequence is designed to produce secreted or membrane-anchored proteins [22] (Figure1). Vaccines 2021, 9, x FOR PEER REVIEW 3 of 15 Vaccines 2021, 9, 61 3 of 14 Figure 1. Simplified overview of the events that follow uptake of an LNP-formulated mRNA vaccine by a cell. Once the mRNAFigure 1. molecule Simplified is released overview from of the the events LNP into that the follow cytosol, uptake it is of sensed an LNP-formulated by toll-like receptors mRNA (TLR), vaccine e.g., by TLR3 a cell. or Once 7/8 andthe bymRNA retinoic molecule acid-inducible is released gene from (RIG)-I, the LNP which into the promotes cytosol, secretion it is sensed oftype by toll-like I interferons receptors (IFNs) (TLR), to the e.g. extracellular TLR3 or 7/8 and matrix by retinoic acid-inducible gene (RIG)-I, which promotes secretion of type I interferons (IFNs) to the extracellular matrix that that will create a milieu that favors Th1 responses over Th2. mRNA is directly translated by ribosomes into polypeptides will create a milieu that favors Th1 responses over Th2. mRNA is directly translated by ribosomes into polypeptides which which are processed by the proteasome system, leading